1. Field of the Invention
The present invention relates to the treatment and prevention of diseases that are primarily due to T cell immune responses. In particular, it relates to the suppression or elimination of certain autoimmune diseases, graft rejection, and allergic disorders by treatment with interleukin-4 (IL-4) and the specific antigen involved, thus allowing the killing of only the subpopulation of T cells that recognizes this specific antigen. In this manner, IL-4 pretreatment sensitizes T cells to undergo programmed cell death following T cell receptor engagement.
2. Description of Related Art
Apoptosis is a form of programmed cell death that occurs in many biological systems (1-5). An apoptotic cell undergoes a specific program of events dependent upon active metabolism that contributes to its own self-destruction. Distinct morphological changes occur during this process such as membrane blebbing and cytoplasmic and nuclear condensation. These changes are accompanied by fragmentation of genomic DNA into pieces constituting one to several nucleosomes. In the final stages, the cell disintegrates into apoptotic bodies that are specifically recognized and phagocytozed by neighboring cells.
T lymphocytes are sensitive to apoptotic cell death induced by a variety of stimuli at multiple points in their lifespan. Experimental evidence strongly suggests that programmed cell death normally plays a large role in shaping and maintaining the T cell repertoire. Repertoire here is defined by the number of distinct antigen receptor specificities contained in the entire pool of T lymphocytes in the organism. Each T lymphocyte bears surface receptors for antigen that are all of identical structure on that cell and therefore are said to represent a single antigen specificity. Since each T cell has a unique specificity, the total collection of antigen specificities in an organism is the sum of different individual T cells, thus the T cell repertoire. By eliminating or expanding the number of individual T cells, the responsiveness of an organism to a particular antigen can be either curtailed or enhanced, respectively. These changes have been documented to occur and are known as changes in the T cell repertoire. Alterations in the T cell repertoire occur naturally during T cell development such that only a small fraction of thymocytes (or immature T cells) survive the intrathymic development and selection events that allow emigration of developing T cells to the peripheral circulation (6,7). The majority of thymocytes appear to undergo apoptotic cell death in the thymus because they bear particular receptors. This "editing" of the T cell repertoire is thought to be the result of two processes: lack of positive selection, and negative selection or clonal deletion. The latter is fundamental to the establishment of self-tolerance as cells expressing potentially autoreactive receptors are actively eliminated. Fetal thymic organ culture (8), in vivo (9), and in vitro (10,11) experiments have shown that the double positive (CD4.sup.+,CD8.sup.+) thymocytes appear to be more sensitive to apoptotic death induced by T cell receptor occupancy than more mature single positive cells. These double positive cells are also sensitive to programmed cell death induced by glucocorticoids (12).
Transformed T cells undergo activation-induced death from stimuli that are normally mitogenic for T cells (13-19). These include antigen, anti-TCR or CD3 mAb binding, the combination of phorbol ester and Ca.sup.2+ ionophore, and mAb modulation of alternative activation molecules Thy-1 and Ly-6. These cells are also susceptible to glucocorticoid-induced apoptosis. The processes of activation- and glucocorticoid-induced programmed cell death are mutually antagonistic in transformed T cells (20-22).
Mature untransformed T cells have been shown to undergo apoptosis in response to various stimuli, such as IL-2 deprivation in the case of cells requiring IL-2 for viability (23), and modulation of the Fas antigen by the APO-1 mAb (24,25). Additionally, it has recently been demonstrated that IL-2 programs mature T lymphocytes to undergo apoptosis in response to antigen receptor stimulation both in vitro and in vivo (26). T cells must be under the influence of IL-2 prior to T cell receptor stimulation for apoptosis to occur, and the amount of cell death rises with increased amounts of IL-2. This process is selective, such that only stimulated T cells triggered by their specific antigen receptor and not by bystander cells undergo cell death. This apparent feedback pathway, termed propriocidal regulation, may represent a mechanism by which T cell responses are regulated (26).
The discovery that interleukin-4 (IL-4) predisposes T lymphocytes to programmed cell death, or apoptosis, allows for a novel method of therapeutic intervention in disease processes in humans and animals primarily caused by the action of IL-4-responsive T cells (27). In essence, this involves specifically inducing the death of a subpopulation of T lymphocytes that are capable of causing disease, while leaving the majority of T lymphocytes substantially unaffected. This method of intervention contrasts with, and is potentially far superior to, currently used therapeutic methods that cause a general suppression or death of T lymphocytes. Examples of widely-used general immunosuppressive agents are corticosteroids, such as prednisone, which are used to treat autoimmune diseases and allergic conditions, and cyclosporin A, which is used for treating graft rejection (28). These treatments suffer from the drawback of severely compromising immune defenses, by debilitating a large portion, if not the entire T cell repertoire. This leaves the patient vulnerable to infectious diseases. The two key elements of the present process are that: i) only the subset of T cells that reacts with antigens that cause the disease are affected by the treatment; and ii) the T cells affected by the treatment are killed, i.e., they are permanently removed from the repertoire.
Several general principles underlie the present process. T cells recognize antigen in the form of short peptides that form noncovalent complexes with major histocompatibility complex (MHC) proteins on the surface of antigen-presenting cells found throughout the body (29). Antigens may also take the form of polysaccharides, organic molecules, or nucleic acids. Each T cell bears a unique antigen receptor called the T cell receptor (TCR) that is capable of recognizing a specific antigen-MHC complex. Through rearrangement of the gene segments containing the protein-coding segments of the TCR, a vast array, perhaps a virtually unlimited number of combinations, of different TCRs are generated (30). By a mechanism termed "allelic exclusion", each T cell bears a single unique TCR. The T cell repertoire is therefore a large number of T cells, each with a distinct TCR that recognizes a specific antigen-MHC complex. It is this vast array of T cells that allows immunological responses to the diversity of antigenic structures on invading micro-organisms, tumor cells, and allografts, thus preserving the integrity of the organism.
Most antigens are able to elicit a response in only a very tiny fraction of the T cell repertoire (31). For example, the initial response to protein antigens may involve as few as 1 in 1000 to 1 in 10,000 T lymphocytes (32). For this reason, diseases caused by T cell reactivity are mediated by only a small subset of the large repertoire of T cells (33). In particular, in those cases where it has been directly measured, such as in multiple sclerosis, the fraction of the T cell repertoire which mediates disease is quite small (33). The important feature of the T cell subset that participates in disease is that it involves T cells which specifically recognize an antigen that provokes the disease. In allergic conditions, the antigen causes the release of inflammatory response molecules. For example, "helper" T cells secrete lymphokines such as IL-4 that cause B cells to produce the inflammatory antibody IgE. In autoimmune diseases, the antigen may be derived from a specific organ in the body and, when recognized by a subset of T cells, stimulates the T cells to attack that organ. A similar effect occurs during graft rejection. Antigenic proteins in the transplanted organ evoke a response in a subset of T cells that attacks the engrafted tissue. For unknown reasons, the fraction of T cells recognizing foreign or "allo" tissue is significantly higher than the number that will typically recognize a protein antigen. Nonetheless, the number of responding T cells is still a distinct minority (1-10%) of the overall T cell repertoire (34).
In a typical T cell response to a specific antigen-MHC complex, stimulation of the TCR (35) results in a cascade of gene activation events. These have been extensively characterized at the molecular level, and two such activation events are especially germane to the present invention i) production of growth lymphokines such as IL-4 and ii) expression of the cell surface proteins that constitute high-affinity receptors for IL-4. Resting T cells express small numbers of high affinity IL-4 receptors; this number increases following activation (36). IL-4 is a 15,000 dalton protein that causes T cells bearing the appropriate high affinity receptor to divide (37,38). The production of IL-4 followed by its interaction with its receptor causes an autocrine mechanism that drives the T cells into the cell cycle. This leads to an initial expansion of T cells that are specifically reactive with the antigen. At present, evidence indicates that in both the human and murine immune systems, a subclass of T lymphocytes called CD4.sup.+ T.sub.H 2 cells may proliferate after antigen activation by producing and responding to IL-4 (39). This subset plays an important and perhaps unique role in stimulating B cells to produce immunoglobulin (Ig) (40). This is because IL-4 and other lymphokines produced by T.sub.H 2 cells, such as IL-5 and L-6, act as differentiation factors for B cells that are crucial for Ig production. Therefore, in autoimmune diseases in which Ig plays a pathogenetic role, the elimination of CD4.sup.+ T.sub.H 2 lymphocytes represents a highly effective way to halt disease. Our results and those of others (27,40-43) show that other classes of T cells including CD4.sup.+ T.sub.H 1 type lymphocytes that mediate delayed-type hypersensitivity as well as CD8.sup.+ cells that mediate cytotoxicity will also proliferate in response to IL-4 and are predisposed to TCR induced apoptosis. Therefore, IL-4 has a potentially broad role in T cell growth during immune responses. Thus, IL-4 could be broadly active in different classes of T cells to predispose them to apoptosis. The present inventive discovery indicates that IL-4 also has the surprising effect of predisposing the expanded pool of either human or mouse T cells to apoptosis or programmed cell death if they are again stimulated or rechallenged through the TCR (27). In the present work described supra, the degree of apoptosis achieved in T cells is correlated positively with both the level of IL-4 the cells experience during their initial expansion, the strength of the TCR stimulation upon rechallenge, and the timing of the rechallenge. In lymphokine-predisposed apoptosis, the effects wear off 2-3 days after lymphokine is no longer present, hence rechallenge must occur within that period (44). The process of activation and apoptosis eventually depletes the antigen-reactive subset of the T cell repertoire.
Apoptosis is a type of programmed cell death in which the T cell nucleus shrinks, the genetic material (DNA) progressively degrades, and the cell collapses (1-5). Evidence suggests that cells cannot recover from apoptosis, and that it results in irreversible killing (1-5). T cells that do not undergo apoptosis but which have become activated will carry out their "effector" functions by causing cytolysis, or by secreting lymphokines that cause B cell responses or other immune effects (45). These "effector" functions are the cause of tissue damage in autoimmune and allergic diseases or graft rejection. A powerful approach to avoiding disease would therefore be to permanently eliminate by apoptosis only those T cells reactive with the disease-inciting antigens, while leaving the majority of the T cell repertoire intact. Apoptosis and T cell deletion caused by antigenic stimulation have been demonstrated in model systems, but since a mechanism for this phenomenon was not previously known, it was not possible to use this in a therapeutically effective way (46-50).
By using IL-4 as an agent that predisposes T cells to death by TCR stimulation in appropriate cycle with immunization with the antigen(s) leading to autoimmune disease or graft rejection, the death of disease-causing T cells can be invoked. Specific methods are described for i) treatment of autoimmune or allergic diseases by identified protein antigen and IL-4, and ii) treatment of graft rejection by blood cell antigens and IL-4. Such methods, by logical extension, can be further developed for other diseases of man or animals that result from the effects of T cells activated by specific antigens. Because the vast majority of immune responses depend on T cell activation, it is predicted that this form of therapy could be applied to a wide variety of autoimmune and allergic conditions especially where antibody production is involved (51,52).
In several human autoimmune diseases, data have indicated that antigen-activated T cells play a key role in the production of disease. These include but are not limited to: 1) multiple sclerosis (53-58); 2) uveitis (59,60); 3) arthritis (61-63); 4) Type I (insulin-dependent) diabetes (64,65); 5) Hashimoto's and Grave's thyroiditis (66-68); and 6) autoimmune myocartiditis (69). The ethical limits on human experimentation have made it very difficult to prove that T reactivity is the sole inciting agent of these diseases. Nonetheless, a large body of experimental work on animal models--murine experimental allergic encephalitis as a model for multiple sclerosis (70,71), BB diabetic rats for human diabetes (72,73), murine collagen-induced arthritis for rheumatoid arthritis (74,75), and S antigen disease in rats and guinea pigs for human autoimmune uveitis (76, 77), among others--suggests that T cells are the critical agent of these diseases. From recent work, the identity of disease-causing proteins or peptide antigens is emerging: i) multiple sclerosis: the peptide epitopes of myelin basic protein (MBP) residues 84-102 and 143-168 (54,57,78,79); ii) autoimmune uveitis: the human S antigen, which has been recently molecularly cloned (59, 80); iii) type II collagen in rheumatoid arthritis (81); and iv) thyroglobulin in thyroiditis (82). Similarly, a wide variety of proteins have been identified which stimulate the production of the allergic immunoglobulin IgE, which is the underlying immunological reaction for common allergies. IgE is produced by B lymphocytes in a process that requires lymphokines produced by antigen-activated T cells known as "T cell help". The class of CD4.sup.+ "helper" T cells that stimulate B cells (T.sub.H 2 cells) typically produce and respond to IL-4 (39,40).
The basic concept of the present therapeutic approach is very simple. Disease-causing T cells are first challenged by immunization to cause the activated T cells to express high affinity IL-4 receptors and, for T.sub.H 2 cells, to begin producing and secreting IL-4. When the cells are expressing high levels of IL-4 receptor, additional human IL-4 is infused to very efficiently drive all the activated cells into the cell cycle. The cells under the influence of IL-4 are then caused to undergo apoptosis by re-immunization with antigenic peptide or protein. Further, if the antigen is capable of stimulating sufficient IL-4 production, it may not be necessary to administer exogenous IL-4. In either case, the timing of rechallenge is important--it must occur within a short interval such as 2-3 days after the first stimulus when cells bear high levels of the IL-4 receptor and are responding to exogenous or endogenous IL-4.
The conceptual advance provided by the inventive discovery that underlies the present methods is that T cell immunity works as a balance between the production and destruction of antigen-specific T lymphocytes. Previously, investigators have focused on the use of lymphokine growth factors such as IL-4 to increase the proliferation and responsiveness of T lymphocytes (38,43,83-87). It is now proposed that the opposing T cell mechanisms be used therapeutically. The discovery that IL-4 predisposes T cells to death is contrary to the previously understood properties of IL-4, and provides a radically new approach to the treatment of diseases caused by T cell reactivity. By providing physicians and medical researchers with the basis of the present inventive discovery, the processes of immune autoregulation leading to T cell destruction can be exploited in combatting disease.
It has been previously known for some time that prior activation and lymphokine production were capable of diminishing immune responsiveness both in vivo and in vitro (46-48). The mechanisms underlying these effects were not understood. Absent the knowledge that IL-4 predisposes T lymphocytes to antigen-dependent apoptosis, it was not possible to manipulate this phenomenon for medical or therapeutic purposes. It is now possible to rigorously study the kinetics and dose requirements of IL-4 in the predisposition phase, and antigen in the apoptosis phase, to routinely optimize the treatment cycle for a given disease following the guidance provided herein.
That this process depends on the discovery of a novel property of IL-4 is particularly auspicious. IL-4 has been thoroughly studied since its discovery in 1982 (85,86). It is well-understood genetically, its cDNA and gene have been molecularly cloned, and antibodies against the protein for immunodetection have been prepared (87,88). IL-4 is already available pharmaceutically in a form for use in humans and studies in human cancer victims have given insights into how IL-4 affects human physiology at different doses (89-92). All of these features significantly enhance the feasibility of its novel use to cause auto-destruction of disease-causing T lymphocytes for the treatment of a wide variety of diseases in humans and other mammals.